Skip to main content
SpringerLink
Log in

Measurement of chlorophylls a and b and bacteriochlorophyll a in organisms from hypereutrophic auxinic waters

  • 8th Asian Pacific Phycological Forum
  • Published:
Journal of Applied Phycology Aims and scope Submit manuscript

Abstract

Sewage lagoons and wastewater ponds from industrialised swine and poultry farms are typically hypereutrophic, auxinic and dominated by purple non-sulphur bacteria and unicellular green algae both typically growing photoheterotrophically. To manage such ponds, it is essential to know the balance between oxygenic and anoxygenic photosynthesis. Typical spectrophotometric algorithms to estimate chlorophyll use 750 nm as a zero (A750 nm) but a 750 nm zero protocol is unsuitable where substantial amounts of bacteriochlorophylls are present. Algorithms were developed to estimate chlorophylls a and b (Chl a and Chl b) and bacteriochlorophyll a (BChl a) in solvent in ethanol, 7:2 acetone/ethanol and 90% acetone. The algorithms use an 850-nm absorbance zero (A850 nm) well outside the absorbance ranges of both chlorophylls and bacteriochlorophylls in solvent. There are many habitats where the presence of anoxygenic photosynthetic bacteria is unsuspected and so using a routine A750 nm zero effectively masks their presence and leads to underestimations of Chl a and Chl b. The in-solvent red peak of bacteriochlorophyll c is too close to that of Chl a for BChl c and Chl a to be resolved spectroscopically, but its presence can be easily identified from in vivo scans. The spectroscopic advantage of 90% acetone is negated by its poor quantitative extraction of pigments. Acetone/ethanol (7:2) is an excellent solvent spectroscopically and as an extractant.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Allen MM (1968) Simple conditions for growth of unicellular blue-green algae on plates. J Phycol 4:1–4

    Article  CAS  Google Scholar 

  • Belila A, Abbas B, Fazaa I, Saidi N, Snoussi M, Hassen A, Muyzer G (2013) Sulfur bacteria in wastewater stabilisation ponds periodically affected by the ‘red-water’ phenomenon. Appl Microbiol Biotechnol 97:379–394

    Article  CAS  Google Scholar 

  • Blankenship RE, Madigan MT, Bauer CE (1995) Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht

    Book  Google Scholar 

  • Chandaravithoon P, Nakphet S, Ritchie RJ (2017) Photosynthesis in a sewage pond. 8th Asian Pacific Phycological Forum, Pullman Hotel, Kuala Lumpur, Malaysia, 8–13 October 2017 (Abstract CS-12-2, p 160)

  • Clayton RK (1966) Spectroscopic analysis of bacteriochlorophylls in vitro and in vivo. Photochem Photobiol 5:669–677

    Article  CAS  Google Scholar 

  • Clayton RK, Clayton BJ (1981) B850 pigment-protein complex of Rhodopseudomonas sphaeroides: extinction coefficients, circular dichroism, and the reversible binding of bacteriochlorophyll. Proc Nat Acad Sci USA 78:5583–5587

    Article  CAS  Google Scholar 

  • Cohen-Bazire G, Sistrom WR, Stanier RY (1957) Kinetic studies of pigment synthesis in non-sulfur purple bacteria. J Cell Comp Physiol 49:25–68

    Article  CAS  Google Scholar 

  • Díez B, Bauer K, Bergman B (2007) Epilithic cyanobacterial communities of a marine tropical beach rock (Heron Island, Great Barrier Reef): diversity and diazotrophy. Appl Environ Microbiol 73:3656–3668

    Article  Google Scholar 

  • Falkowski PG, Raven JA (2007) Aquatic photosynthesis, 2nd edn. Princeton University Press, Princeton, NJ, USA

    Google Scholar 

  • Gitelson A, Stark R. Oron G, Dor I (1997) Monitoring of polluted water bodies by remote sensing. In: Remote sensing and geographic information systems for design and operation of water resources systems, proceedings of Rabat symposium S3, April 1997. IAHS Publ. No 242, pp 181–188

  • Gitelson A, Stark R, Dor I, Michielson O, Yacobi YZ (1999) Optical characteristics of the phototroph Thiocapsa roseopersicina and implication for real time monitoring of the bacteriochlorophyll concentration. Appl Environ Microbiol 65:3392–3397

    CAS  PubMed  PubMed Central  Google Scholar 

  • Goericke R (2002) Bacteriochlorophyll a in the ocean: is anoxygenic bacterial photosynthesis important? Limnol Oceanogr 47:290–295

    Article  CAS  Google Scholar 

  • Hubas C, Jesus B, Passarell C, Jeanthon C (2011) Tools providing new insight into coastal anoxygenic purple bacterial mats. Res Microbiol 162:858–868

    Article  Google Scholar 

  • Kiang NY, Siefert J, Govindjee, Blankenship RE (2007) Spectral signatures of photosynthesis. I. Review of earth organisms. Astrobiology 7:222–251

    Article  CAS  Google Scholar 

  • Kim M-K, Harwood CS (1991) Regulation of benzoate-CoA ligase in Rhodopseudomonas palustris. FEMS Microbiol Lett 83:199–203

    CAS  Google Scholar 

  • Kim JK, Lee B-K (2000) Mass production of Rhodopseudomonas palustris as diet for aquaculture. Aquac Eng 23:281–293

    Article  Google Scholar 

  • Kim MK, Choi K-M, Yin C-R, Lee K-Y, Im W-T, Lim JH, Lee S-T (2004) Odorous swine wastewater treatment by purple non-sulfur bacteria, Rhodopseudomonas palustris, isolated from eutrophicated ponds. Biotechnol Lett 26:819–822

    Article  CAS  Google Scholar 

  • Koelsch RK, Chen T-T, Sculte DD (1997) Purple sulphur bacteria in anaerobic treatment lagoons. Nebraska Swine Reports Paper 205:36–38 http://digitalcommons.unl.edu/coopext_swine/205

    Google Scholar 

  • Kolber ZS, Van Dover CL, Niederman RA, Falkowski PG (2000) Bacterial photosynthesis in surface waters of the open ocean. Nature 407:177–179

    Article  CAS  Google Scholar 

  • Larimer FW, Chain P, Hauser L, Lamerdin J, Malfatti S, Do L, Land ML, Pelletier DA, Beatty JT, Lang AS, Tabita FR, Gibson JL, Hanson TE, Bobst C, Torres JLT, Peres C, Harrison FH, Gibson J, Harwood CS (2004) Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nature Biotech 22:55–61

    Article  CAS  Google Scholar 

  • Meckenstock RU, Brunisholz RA, Zuber H (1992) The light-harvesting core-complex and the BS20-subunit from Rhodopseudomonas marina. Part I. Purification and characterisation. FEBS Lett 311:128–134

    Article  CAS  Google Scholar 

  • Namsaraev ZB (2009) Application of extinction coefficients for quantification of chlorophylls and bacteriochlorophylls. Microbiology 78:794–797

    Article  CAS  Google Scholar 

  • Papineau D, Walker JJ, Mojzsis SJ, Pace NR (2005) Composition and structure of microbial communities from stromatolites of Hamelin Pool in Shark Bay, Western Australia. Appl Environ Microbiol 2005:4822–4832

    Article  CAS  Google Scholar 

  • Porra RJ (1990) A simple method for extracting chlorophylls from the recalcitrant alga, Nannochloris atomus, without formation of spectroscopically-different magnesium-rhodochlorin derivatives. Biochm Biophys Acta 1019:137–141

    CAS  Google Scholar 

  • Porra RJ (1991) Recent advances and reassessments in chlorophyll extraction and assay procedures for terrestrial, aquatic and marine organisms, including recalcitrant algae. In: Scheer H (ed) Chlorophylls. CRC Press, Boca Raton, pp 31–57

    Google Scholar 

  • Porra RJ (2006) Spectrophotometric assays for plant, algal and bacterial chlorophylls. In: Grimm B, Porra RJ, Ruediger W, Scheer H (eds) Chlorophylls and bacteriochlorophylls: biochemistry, biophysics, functions and applications. Springer, Dordrecht, pp 95–106

    Chapter  Google Scholar 

  • Porra RJ (2011) A proven simultaneous equation assay for chlorophyll a and b using aqueous acetone and similar assays for recalcitrant algae. In: Roy S, Llewellyn CA, Egeland ES, Johnsen G (eds) Phytoplankton pigments: characterisation, chemotaxonomy and applications in oceanography, SCOR-UNESCO 2011, Appendix 8A. Cambridge University Press, pp 366–371

  • Ritchie RJ (2006) Consistent sets of spectrophotometric equations for acetone, methanol and ethanol solvents. Photosynth Res 89:27–41

    Article  CAS  Google Scholar 

  • Ritchie RJ (2008) Universal chlorophyll equations for estimating chlorophylls a, b, c & d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol or ethanol solvents. Photosynthetica 46:115–126

    Article  CAS  Google Scholar 

  • Ritchie RJ (2013) The use of solar radiation by a photosynthetic bacterium, Rhodopseudomonas palustris: model simulation of conditions found in a shallow pond or flatbed reactor. Photochem Photobiol 89:1143–1162

    Article  CAS  Google Scholar 

  • Ritchie RJ, Larkum AWD (2013) Modelling photosynthesis in shallow algal production ponds. Photosynthetica 50:481–500

    Article  Google Scholar 

  • Ritchie RJ, Mekjinda N (2015) Measurement of photosynthesis using PAM technology in a purple sulphur bacterium Thermochromatium tepidum (Chromatiaceae). Photochem Photobiol 91:350–358

    Article  CAS  Google Scholar 

  • Ritchie RJ, Runcie JW (2013) Measurement of the photosynthetic electron transport rate in an anoxygenic photosynthetic bacterium Afifella (Rhodopseudomonas) marina using PAM Fluorometry. Photochem Photobiol 89:370–383

    Article  CAS  Google Scholar 

  • Siefert E, Irgens RL, Pfennig N (1978) Phototrophic purple and green bacteria in a sewage treatment plant. Appl Environ Microbiol 35:38–41

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sletten O, Singer RH (1971) Sulfur bacteria in red lagoons. J Water Pollut Control Fed 43:2118–2122

    CAS  Google Scholar 

  • Takahashi M, Ichimura S (1970) Photosynthetic properties and growth of photosynthetic sulfur bacteria in lakes. Limnol Oceanogr 15:929–944

    Article  CAS  Google Scholar 

  • van Niel CB (1944) The culture, general physiology, morphology and classification of the non-sulphur purple and brown bacteria. Bact Rev 8:1–118

    PubMed  Google Scholar 

  • van Niel CB (1971) Techniques for the enrichment, isolation and maintenance of the photosynthetic bacteria. Methods Enzymol 23:3–28

    Article  Google Scholar 

  • Yurkov VV, Beatty JT (1998) Aerobic anoxygenic phototrophic bacteria. Microbiol Mol Biol Rev 62:695–724

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang D, Yang H, Huang Z, Zhang W, Liu S-J (2002) Rhodopseudomonas faecalis sp. nov., a phototrophic bacterium isolated from an anaerobic reactor that digests chicken faeces. Intl J Syst Evol Microbiol 52:2055–2060

    CAS  Google Scholar 

Download references

Acknowledgements

The author wishes to thank Prince Songkla University—Phuket for providing facilities for the project. The co-operation of Phuket Integrated Waste Management (Wichit Sub-district, Mueang Phuket, District, Phuket 83000, Thailand) in encouraging this study and allowing us to collect sewage pond water samples is gratefully acknowledged. My Masters student, Piamsook Chandaravithoon assisted me in collecting the sewage pond samples. The author would like to give special thanks to Dr. Robert J Porra (CSIRO, Plant Industry, Canberra) for his valuable comments on the manuscript. Some of the data presented here was used as part of an oral presentation (Chandaravithoon et al. 2017: Photosynthesis in a sewage pond) at the recent 8th Asian Pacific Phycological Forum at the Pullman Hotel, Kuala Lumpur, Malaysia, 8–13 October 2017 (CS-12-2).

Funding

The project was partially funded by the Faculty of Technology and Environmental Science, Prince Songkla University-Phuket.

Author information

Authors and Affiliations

  1. Tropical Environmental Plant Biology Unit, Faculty of Technology and Environment, Prince of Songkla University—Phuket Campus, Phuket, 83120, Thailand

    Raymond J. Ritchie

Authors
  1. Raymond J. Ritchie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raymond J. Ritchie.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ritchie, R.J. Measurement of chlorophylls a and b and bacteriochlorophyll a in organisms from hypereutrophic auxinic waters. J Appl Phycol 30, 3075–3087 (2018). https://doi.org/10.1007/s10811-018-1431-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-018-1431-4

Keywords

Search

Navigation